This is a continuation of a project to analyze, annotate, organize, and integrate RNA 3D structural data with RNA sequence and functional data and deliver it to biomedical researchers. The significance of the proposal lies in the prominent role of RNA, much of it non-protein-coding and structured, in a host of cellular and physiological processes, as revealed by recent high-throughput studies. Thousands of new non-coding RNAs have been discovered, many of which are expressed with temporal and spatial specificity. RNA plays many different roles in the human body, from carrying genetic information and regulating gene expression, to mediating intra-cellular communication, responding to environmental signals, and catalyzing important cellular processes such as protein synthesis. Deciphering the relationships between 3D structure and sequence is key to understanding RNA function. RNA 3D motifs are vital components in the architecture of RNA and in RNA- RNA and RNA-protein interactions. We are developing tools to study the variability of 3D motif structures across different conditions within the same and among homologous RNA molecules, integrating 3D structure and sequence data. These tools will be key to improving our ability to predict structure from sequence. The specific aims of the project are: 1. Deepen our understanding of RNA 3D structure by extracting and organizing 3D motifs. 2. Improve our understanding of the structural variation of RNA molecules and RNA complexes by examining and comparing 3D structures assigned to the same sequence/structure equivalence class. 3. Accelerate the study of the correspondence between RNA 3D structure and RNA sequence. 4. Provide annotations for RNA-protein interactions. 5. Develop new and extended tools for delivery, search and visualization of the RNA sequence and structure annotations. In the previous grant period, we created the RNA 3D Motif Atlas and a data pipeline to automatically extract, analyze and cluster RNA hairpin and internal loop motifs from new RNA-containing 3D structures on a monthly schedule; in the current project, we will do the same for RNA-RNA interaction motifs and RNA multi-helix junctions. We will use real-space refinement (RsR) statistics to evaluate, at the nucleotide level, how well RNA 3D structures fit the underlying experimental x-ray data. We will create and integrate annotations of RNA-protein interactions from a variety of resources; RNA- protein interactions are a key way that regulatory RNAs carry out their functions in living organisms. Finally, all products of the research will be made publicly available through the Nucleic Acid Database (NDB) website. These include interactive 2D maps and 3D visualizations of large RNAs, services to allow geometric searches for 3D structure fragments, and the many new annotations. These services will benefit a wide variety of biomedical researchers who will find ready access to what is known about the 3D structures of RNA motifs, interactions they make with other RNAs, interactions they make with proteins, and how to identify these through RNA sequence alone.